The dynamic microtubule cytoskeleton mediates intracellular organization, is responsible for force generation in dividing or migrating eukaryotic cells, and forms tracks for intracellular trafficking. The key dynamic properties of microtubules, including polarized growth and dynamic instability, stem directly from the unique structure and GTP hydrolysis activity of their building blocks, asymmetric dimers of ?- and ?-tubulin. Active ??-tubulin dimers are assembled, maintained at high concentration in the cytoplasm, and degraded by highly conserved tubulin cofactors whose molecular mechanisms remain mostly mysterious, due in part to a lack of biochemical reconstitution. In addition, we do not understand how conserved families of Tumor Overexpressed Gene (TOG) domain proteins manipulate ??-tubulin dimer conformation and incorporation to modulate microtubule dynamics. The importance of these pathways is underscored by the fact that genetic defects that impair the regulation of soluble tubulin are linked to inherited neurological and developmental disorders. This proposal is focused on understanding the mechanisms of these soluble ??-tubulin regulators and how they impact microtubule dynamics. Our biophysical approach combines methods across multiple resolution scales, including in vitro reconstitution of purified protein complexes, 3D structural determination by x-ray crystallography and electron microscopy, and fluorescence microscopy-based assays of microtubule dynamics in real time. First, we will determine the mechanism by which tubulin cofactors regulate ??-tubulin dimer assembly, activation, and degradation. We propose a new model, based on extensive biochemical reconstitution and structural studies, in which tubulin cofactors and a dedicated Arf-like G-protein form multi-subunit platforms on which soluble ??-tubulins are manipulated, powered by GTP hydrolysis cycles. To test this model we will; 1) determine the 3D structures of tubulin cofactor platforms in three biochemical states to understand their molecular organization and conformational changes during catalysis; 2) dissect the mechanism of GTP hydrolysis during catalytic regulation; and 3) follow up exciting preliminary studies showing their direct effect in activating microtubule polymerization in vitro. Second, we will examine the molecular mechanisms of two types of TOG domains found in two classes of microtubule regulators, XMAP215/Dis1 and CLASP, which despite their structural similarity perform different functions. To determine how these proteins control microtubule assembly and function, we propose to explore multiple hypotheses, including a model based on our preliminary studies suggesting a wrapped organization of multiple TOG domains around each ??-tubulin dimer. To test this model we will: 1) determine 3D structures of multi-TOG- domains-soluble ??-tubulin dimer complexes using electron microscopy; 2) determine x-ray structures for two- adjacent TOG domains sets, from the two classes, in complex with soluble ??-tubulin dimer; 3) dissect the unique functions of TOG domains from the two regulator classes by studying chimeric proteins, generated by domain swapping, in microtubule dynamics assays in vitro; and 4) visualize multi-TOG domain ??-tubulin complexes at polymerizing microtubule ends. We expect our studies to yield new structural and biophysical models that will provide missing information on the regulation of soluble tubulin activation and recruitment during microtubule polymerization. This understanding will in turn point toward new strategies for addressing defects in tubulin biogenesis and regulation, potentially impacting patients with a range of developmental and neurological disorders.